Secreted and membrane proteins undergo folding and other post-translational modifications in the endoplasmic reticulum (ER)-Golgi system. Disruption of the homeostasis of this system causes cellular stress that can lead to apoptosis. ER homeostasis can be altered by changes in Ca2+ concentration or redox status, altered glycosylation, or accumulation of unfolded or misfolded proteins in the ER lumen. To overcome stress, the secretory system has evolved an adaptive stress response mechanism known as the unfolded protein response (UPR). Activation of the mammalian UPR results in at least three responses: (1)′ the amount of new protein translocated to the ER lumen is reduced through reduction in translation; (2) accumulated protein in the ER lumen is retrotranslocated to the cytosol and degraded; and (3) the ER-Golgi secretory system is remodeled so that the protein folding and processing capacities in the system are enhanced.
The capacity enhancement of the ER-Golgi system in response to stress involves upregulation of folding and processing enzymes. These enzymes include ER chaperones, enzymes involved in glycosylation and disulfide bond formation, and enzymes participating in vesicle transportation. In mammalian cells, IRE1 and ATF6 proteins are the major transducers of this branch of the UPR pathway. IRE1 protein is an ER transmembrane glycoprotein with kinase and endonuclease activities at its C-terminal cytosolic domain. At least two IRE1 genes have been identified in mice, IRE1α and IRE1β. IRE1α is essential for viability and is broadly expressed. IRE1β has been detected only in the gastrointestinal mucosa. ER stress leads to oligomerization of IRE1 proteins and trans-autophosphorylation of their cytosolic domains. Phosphorylation of IRE1 activates its endonuclease activity which excises an intron from the mRNA of the transcription factor X-box binding protein 1 (XBP1). This splicing event results in the conversion of a transcription-inactive XBP1 isoform (i.e., XBP1u) to a transcription-active XBP1 isoform (i.e., XBP1s or XBP1p). XBP1p then travels into the nucleus, where it binds to its target sequences including ER stress response element (ERSE) and UPR element (UPRE), in the regulatory regions of ER-Golgi chaperone/enzyme genes, to induce their transcription. Many UPR target genes have one or more copies of ERSE or UPRE sequence in their promoter regions.
ATF6 (activating transcription factor 6) is another ER transmembrane protein. ER stress leads to the transit of ATF6 protein to the Golgi compartment where its cytosolic domain is cleaved by Site 1 and Site 2 proteases. The cleaved cytosolic domain travels to the nucleus and acts as a transcription factor by binding to ERSE sequences, which in turn up-regulates a variety of chaperones and processing enzymes in the secretory pathway.
Activation of the UPR pathway in mammalian cells also leads to a transient inhibition of protein translation through the PERK signaling pathway. PERK is an ER transmembrane kinase which can phosphorylate the eukaryotic translation initiation factor eIF2α in response to ER stress. Phosphorylation of eIF2α prevents the assembly of the 43S ribosomal pre-initiation complex and therefore results in translation attenuation. Paradoxically, phosphorylation of eIF2α also results in rapid synthesis of transcription factor ATF4, which in turn enhances the expression of a proapoptotic transcription factor CHOP. CHOP potentiates cell death when the detrimental effects of ER stress can no longer be overcome.
Overexpression of secreted recombinant proteins in mammalian cells often leads to low production yield. A commonly used method to improve protein production is to increase the transcriptional rates, such as using stronger promoters or increasing gene copy numbers. However, increased transcriptional rates may exacerbate ER stress and, therefore, often fails to significantly improve the yield. In some cases, it may even further reduce the production yield.